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A specific methylation detection primer and endometriosis diagnostic kit

A diagnostic kit and endometrium technology, applied in biochemical equipment and methods, microbiological determination/testing, DNA/RNA fragments, etc., can solve problems affecting diagnostic accuracy and sensitivity, false positives, and endometriosis The accuracy and sensitivity of positional symptoms are not high enough, so as to achieve the monitoring of prognostic goals, improve pregnancy outcomes, and improve the quality of life.

Active Publication Date: 2021-03-09
PEKING UNIV FIRST HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the accuracy and sensitivity of diagnosing endometriosis through the methylation level of the SF-1 promoter region are not high enough. On the one hand, it has been found that in a small number of endometriosis patients, SF-1 starts There is still a certain methylation state in the subregion; on the other hand, the current conventional method for detecting the methylation state is to first modify cytosine with bisulfite, and then perform sequencing to determine the methylation state of a certain site. However, the accuracy and sensitivity of this method are affected by the efficiency of the bisulfite modification reaction, and false positives may occur, thereby affecting the accuracy and sensitivity of the diagnosis

Method used

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  • A specific methylation detection primer and endometriosis diagnostic kit
  • A specific methylation detection primer and endometriosis diagnostic kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1 Kit

[0052] The kit includes: PCR master mix, and:

[0053] The first primer pair: the upstream primer shown in SEQ ID NO.1 and the downstream primer shown in SEQ ID NO.2;

[0054] Second primer pair: upstream primer shown in SEQ ID NO.3 and downstream primer shown in SEQ ID NO.4.

[0055] Wherein, the use conditions of the kit are: 90° C. for 10 minutes, 40 cycles (95° C. for 30 s, 50° C. for 2 minutes, 72° C. for 2 minutes), and 72° C. for 7 minutes.

[0056] The PCR master mix used in the present invention can be any commercially available mixed reagent that can realize DNA PCR amplification, such as AmpliTaq Gold PCR master mix provided by Applied Biosystems.

Embodiment 2

[0057] Example 2 Diagnosis of endometriosis

[0058] 1. Sample collection and DNA extraction

[0059] A small amount of endometriosis lesion tissue was obtained, and endometrial stromal cells were digested, separated and cultured by zymogen method, and the cultured endometrial stromal cells were extracted using a DNA extraction kit. The extraction method was carried out according to the operating instructions of the kit (DNeasy tissue kit, QIAGEN, Valencia, CA).

[0060] 2. Base modification

[0061] Take 500ng DNA for bisulfite modification (according to the kit EZ DNA Methylation Kit, ZymoResearch, Orange, CA operating instructions), the unmethylated cytosine in the DNA is converted into uracil, and the methylated cytosine constant.

[0062] 3. PCR amplification

[0063] Take the above 3ul sodium bisulfite-treated DNA and add it to AmpliTaq Gold PCR master mix (Applied Biosystems) to form a 20ul PCR reaction system for PCR amplification. The primers used are:

[0064] F...

Embodiment 3

[0075] Example 3 Sample detection

[0076] 1. Sample information

[0077] Seven cases of eutopic endometrial tissue from non-endometriosis patients who underwent hysterectomy for uterine fibroids were selected as the control group, and 7 cases of ectopic endometrial tissue from endometriosis patients were selected as the experimental group.

[0078] 2. Sample detection

[0079] Extract the DNA of the experimental group and the control group respectively. The DNA can be extracted using any commercially available kit that can realize DNA extraction. The extraction method is operated according to the instructions of the kit. - Eight clones were tested for endometriosis using the kit in Example 1, that is, the method in Example 2, and the test results are shown in Table 1.

[0080] Table 1 Test results of the experimental group

[0081]

[0082] Wherein, the significance of the score B / A in the table is: A represents the total number of positive clones obtained in each sampl...

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Abstract

The invention discloses a specific methylation detection primer and a diagnostic kit for endometriosis, and relates to the field of biotechnology. According to the SF-1 gene in the endometriosis genome, the present invention designs PCR amplification primers and related kits for endometriosis-specific methylation detection. Use the primers and kit to perform PCR amplification on the sample DNA modified by bisulfite to be tested, obtain the methylation site information of the SF-1 gene according to the sequencing results of the amplified products, and judge according to the methylation site information Whether the sample to be tested is a sample of a patient with endometriosis, so as to realize the diagnosis of endometriosis. The primer or kit provided by the present invention can be used to diagnose endometriosis objectively, sensitively and stably at an early stage, so as to perform timely and targeted treatment and improve the quality of life of women of childbearing age.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a specific methylation detection primer and a diagnostic kit for endometriosis. Background technique [0002] Endometriosis (endometriosis, endometriosis) is a common factor that causes pelvic pain and infertility. Nodules and masses may form. Endometriosis is a hormone-dependent disease mainly seen in women of reproductive age. In recent years, its incidence has been increasing, with an incidence of about 10% among women of reproductive age, however, due to its association with infertility and pelvic pain, its prevalence is significantly higher in these female groups, and Become a common disease in gynecology. According to reports, the prevalence of endometriosis in infertile women is 25%-35%, and that of women with pelvic pain reaches 39-59%. However, 50% of patients with endometriosis have obvious dysmenorrhea, and 30% are combined with infertility, which seriously affects the...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6883C12N15/11
CPCC12Q1/6883C12Q2600/118C12Q2600/156
Inventor 薛晴曾诚吴培莉
Owner PEKING UNIV FIRST HOSPITAL
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